Translational Neurodegeneration

Extracellular amyloid plaques, the pathognomonic hallmark of Alzheimers Disease (AD), are also observed in cognitively unimpaired subjects in the preclinical stages. Progressive accumulation of fibrillar amyloid-{beta} (A{beta}) as plaques and perivascular deposits occur two decades prior to clinical onset, making A{beta} a long-lived peptide. To characterize the amyloid plaques biochemically, both the A{beta}-load as well the post-translational modifications (PTMs) could serve as markers for distinguishing the pre-clinical stage compared to later prodromal and clinical stages of AD. Recently, we described the presence of extensive isomerization of the A{beta} N-terminus in AD post-mortem brains that are significantly increased compared to the age-matched non-AD control brains with A{beta} aggregates in the frontal cortex. In this report, we used targeted mass spectrometry to conduct a quantitative analysis of the most common PTMs associated with A{beta}; pyroglutamation, citrullination, N-terminal truncation (A{beta}4-x), C-terminal truncation (A{beta}42 and A{beta}40), and isomerization of aspartic acid residues (Asp-1 and Asp-7) in postmortem human brain tissue from pathologically negative (no A{beta} plaques) controls, controls with A{beta} plaques, Parkinsons disease (PD) with and without A{beta} accumulation/plaques and symptomatic AD. The AD cases contained statistically significant amounts of Asp-1and Asp-7 isomerized A{beta}1-15 ([~] 90 %) compared to controls (preclinical AD) and PD brains with fibrillar A{beta} aggregates/deposits. We find that ratio of isomerized N-terminus A{beta} (A{beta}1-15) species in the brain detergent soluble pool differentiates older fibrillar A{beta} deposits in symptomatic AD brain compared to A{beta} deposits detected in preclinical AD and PD. Citrullinated A{beta}3pglu-15 was increased only in symptomatic AD, highlighting this A{beta} PTM is a unique feature of parenchymal plaques in advanced AD. Our results have implications for early therapeutic targeting of these modified species as well potential for better biofluid biomarker development for drug efficacy monitoring.

Proteomic studies of human Alzheimers disease brain tissue have exceptional potential to identify protein changes that drive disease and to identify new drug targets. Here, we detail a combined analysis of 38 published Alzheimers disease proteomic studies, generating a comprehensive map of protein changes in human brain tissue across thirteen brain regions, three disease stages (preclinical Alzheimers disease, mild cognitive impairment, advanced Alzheimers disease), and proteins enriched in amyloid plaques, neurofibrillary tangles, and cerebral amyloid angiopathy. Our dataset is compiled into a user-friendly, searchable database called NeuroPro. Our combined analysis included 18,119 reported protein differences in human Alzheimers disease brain tissue, which mapped to 5,311 total altered proteins. Proteomic studies were remarkably consistent. 848 proteins were consistently altered in [≥]5 studies, many of which are understudied in the Alzheimers field. Comparison of protein changes in early-stage and advanced Alzheimers disease revealed significant synapse, vesicle, and lysosomal changes early in disease, but widespread mitochondrial changes only in advanced Alzheimers disease. Comparison of vulnerable and resistant brain regions suggested that protein changes in resistant regions in advanced Alzheimers disease are similar to those in vulnerable regions in early-stage Alzheimers disease, indicating a temporal progression of protein dysfunction during Alzheimers disease advancement. We conclude that NeuroPro is a powerful new resource that provides new insights into human Alzheimers disease brain protein changes and highlights novel proteins of particular interest that may mechanistically drive Alzheimers disease.

BackgroundThiamine diphosphate (TDP) reduction plays an important role in cerebral glucose hypometabolism, the neurodegenerative indicator, in Alzheimers disease (AD). The mechanism underlying TDP reduction remains elusive. Thus, it is critical to define the mechanism and its effect on neurodegeneration, the pathological basis of the disease occurrence and progression. MethodsThe mRNA levels of all known genes associated with thiamine metabolism, including thiamine pyrophosphokinase (TPK), Solute Carrier Family 19 Member 2 (SLC19A2), SLC19A3, and SLC25A19, in brain samples of patients with AD and other neurodegenerative disorders in multiple independent datasets were analyzed. TPK protein levels were further examined in the brain tissues of AD patients and control subjects. A mouse model with conditional knockout (cKO) of TPK gene in the excitatory neurons of adult brain was established. ResultsThe brain TPK mRNA level was markedly lower in AD patients, but not in other neurodegenerative disorders. The brain TPK protein level was also significantly decreased in AD patients. TPK gene knockout in the mice caused cerebral glucose hypometabolism, {beta}-amyloid deposition, Tau hyperphosphorylation, neuroinflammation, and neuronal loss and brain atrophy. Cross-species correlation analysis revealed the similar changes of gene profiling between the cKO mice and AD patients. ConclusionsThe deficiency of brain TPK, a key enzyme for TDP synthesis, is specific to AD. The cKO mice show AD-associated phenotypes and could serve as a new mouse model for AD studies. Our study provides a novel insight into the critical role of TPK in AD pathogenesis and its potential for the disease treatment.

AD presents with severe neurodegeneration which leads to cognitive deficits and dementia. Identifying the molecular signals that attenuate neurodegeneration in AD may be exploited as therapeutic targets. This study revealed that transgenic AD mice (PSAPP) exhibit decreased caveolin-1 (Cav-1), a membrane/lipid raft (MLR) scaffolding protein that organizes synaptic signaling components. Subcellularly, Cav-1 and full length (fl)-TrkB were significantly decreased in MLRs. We thus developed an in vivo gene therapy that re-expresses neuronal-targeted Cav-1 using the synapsin promoter (SynCav1). While AD mice showed significant learning and memory deficits at 9 and 11 months, AD mice that received hippocampal SynCav1 (AD-SynCav1) maintained normal learning and memory at 9 and 11 months respectively. Furthermore, AD-SynCav1 mice showed preserved hippocampal MLR-localized fl-TrkB, synaptic ultrastructure, dendritic arborization and axonal myelin content, all of which occurred independent of reducing amyloid deposit and astrogliosis. Thus, SynCav1 demonstrates translational potential to treat AD by delaying neurodegeneration. SummaryTransgenic PSAPP mice exhibit decreased hippocampal expression of the membrane lipid raft (MLR) scaffolding protein caveolin-1. Synapsin-promoted re-expression of Cav-1 (termed SynCav1) mitigated neuropathology and cognitive deficits. SynCav1 gene therapy has the potential to treat AD and other forms of neurodegeneration.

Familial Alzheimers disease (AD) involving known AD causing genes accounts for a small fraction of cases, the vast majority are sporadic. Neuroinflammation, secondary to viral infection, has been suggested as an initiating or accelerating factor. In this work we tested the hypothesis that SARS-CoV-2 (SCV2) viral infection accelerates the development of AD pathology in mouse models of AD. We profiled transcriptomic changes using transgenic APP/PSEN1 and P301S mouse models that develop AD pathology and k18hACE2 mice that express the humanized ACE2 receptor used by SCV2 to enter cells. This study identified the interferon and chemokine responses constituting key shared pathways between SCV2 infection and the development of AD pathology. Two transgenic mouse models of AD: APP/PSEN1 (develops amyloid pathology) and 3xTg AD (develops both amyloid and tau pathology) were crossed with k18-hACE2 mice to generate hybrid hACE2-3xTg and hACE2-APP/PSEN1 mice. Neuroinflammation and amyloid deposition in the brain of infected mice were imaged in vivo using molecular MRI (mMRI) probes and confirmed postmortem by histopathology. Results show that 11-14-month-old SCV2 infected hACE2-3xTg mice exhibit neuroinflammation 10 days post infection and 4-5-month-old hACE2-APP/PS1 hybrid mice develop amyloid deposits, while age-matched uninfected mice exhibit neither phenotype. This suggests that SCV2 infection could induce or accelerate AD when risk factors are present.

Post-translational modifications (PTMs) in APP and MAPT contribute to plaques and tangles in Alzheimers disease (AD). Yet broader proteome-wide PTMs in the AD brain are relatively unexplored. Therefore, this study highlights associations between PTMs, quantified by mass spectrometry in prefrontal cortex tissue, and Alzheimers disease neuropathology and cognition. Leveraging PTMs quantified from prefrontal cortices in 101 Rush Memory and Aging Project participants. We assessed associations with post-mortem amyloid-{beta} and tau burden, global cognition, and cognitive decline. First, APP and MAPT PTM associations were assessed on these outcomes given their known relevance in AD, followed by assessment of protein-wide effects of PTMs. Then, kinase enrichment analysis was performed on each outcome to assess which kinases might contribute to the results. We observed a novel association of APP-K687 acetylation, a known mutation hotspot driving pathology, with amyloid-{beta} load ({beta}=0.44, P=3.9e-8), while confirming known MAPT PTMs with tangle burden. Further, we identified 20+ novel PTMs associations with AD neuropathology, including ENO2-K256 ubiquitination ({beta}=0.353, P=1.13e-6), PSMD13-K31 ubiquitination ({beta}=0.568, P=1.34e-6), and PLXND1-K1826 ubiquitination ({beta}=0.577, P=7.08e-8) for tangle burden and SYP-K23 ubiquitination ({beta}=1.50, P=4.7e-8), TMEFF2-C80 cysteine oxidation ({beta}=1.64, P=1.1e-8), and STX1B-T121 phosphorylation ({beta}=0.898, P=3.3e-7) for amyloid-{beta} load. Further, kinase enrichment analyses highlight the complexity of disease-related proteome changes with some kinases like CDK5 showing expected over-enrichment (amyloid z=3.44, P=3.0e-4; tau z=4.98, P=3.3e-7) but others like PKC family kinases showing divergent enrichment between amyloid (z=8.98-11.55, P<1.0e-18) and tau (z=-2.83--3.88, P<0.006). This study provides an atlas of brain PTMs within crucial proteins like MAPT and APP and at the proteome-wide level, that impact AD neuropathology and clinical presentation. Further, we explored what kinases might be driving phosphorylation results, emphasizing the complex proteome changes which impact AD. In sum, these results highlight robust post-translational alterations in the AD brain and provide novel targets for future mechanistic studies.

Alzheimers Disease (AD) is distinguished by the presence of two key pathological features: amyloid plaques, accumulations of proteolytic products of Amyloid Precursor Protein, and neurofibrillary tangles (NFTs), intracellular aggregations of microtubule-associated protein tau. NFTs first appear in particular entorhinal cortex (EC) neurons (called pre-alpha neurons) in asymptomatic patients, then continue to spread through other connected brain regions as the disease progresses. This stereotypical progression of tauopathy through synaptically connected brain regions (i.e. Braak stages) not only suggests that the tauopathy in AD spreads transsynaptically, it also raises the question whether particular neuronal subtypes and/or brain regions are especially prone to tauopathy. We explored this question by overexpressing wildtype human tau protein (hTau) in a variety of entorhinal and neocortical neuronal subtypes. We then compared the tendencies of different neuronal cell types to develop different pathological tau species over time and found that tau pathology does indeed develop at markedly different rates in different neuronal subtypes. Perhaps unsurprisingly, the EC is particularly prone, with the likely rat cognates of the pre-alpha neurons (ECLII fan cells) among the first to express pathological tau label. Fan cells were not, however, the neurons with the greatest vulnerability to generate pathological tau species: subsets of ECLIII neurons were found to express disproportional amounts of pathological tau. This is of particular interest given that the next structures to develop AD-related tauopathy after the EC in patients are CA1 hippocampus and subiculum, where ECLIII neurons project, rather than the dentate gyrus and CA3, where ECLII fan cells project. These results demonstrate differential susceptibility of different neuronal subtypes to pathological tau species and suggest distinct roles for different entorhinal neuronal subtypes in the propagation of the tauopathy underlying AD-related neurodegeneration.

Sleep disturbances are prevalent in Alzheimers disease (AD) and Progressive Supranuclear Palsy (PSP), often exacerbating disease progression. Understanding the neuropathological basis of these disturbances is essential for identifying potential therapeutic targets. This study investigates the intermediate nucleus (IntN) of the human hypothalamus--a key sleep-regulating region analogous to the rodent ventrolateral preoptic area (VLPO)--to assess neuronal loss and tau pathology in AD and PSP. Using postmortem brain tissue, we applied unbiased stereology to quantify galanin-expressing neurons and phosphorylated tau (p-tau) accumulation. Among 26 cases analyzed, both AD and PSP exhibited significant neuronal loss in the IntN, with PSP showing the most pronounced reduction (84.9% fewer neurons than healthy controls [HC]). In AD, neuronal loss correlated with Braak staging, with late-stage AD cases (Braak 5-6) demonstrating a 76.9% reduction in galanin-expressing neurons compared to HC, while non-galanin neurons exhibited a more moderate decline (45.7%). In PSP, extensive neuronal loss precluded a clear assessment of p-tau burden. These findings suggest a differential neuronal vulnerability to tau pathology across diseases, aligning with distinct sleep disturbances observed in each condition. PSP, characterized by severe insomnia despite preserved wake-promoting neurons, may be explained by the near-total loss of NREM sleep-regulating neurons. In contrast, AD exhibits a progressive decline in both wake- and sleep-promoting neurons, contributing to excessive daytime sleepiness and sleep fragmentation. This study provides critical insights into the selective neuronal vulnerabilities underlying sleep dysfunction in tauopathies, emphasizing the need for targeted interventions to mitigate sleep disturbances in these disorders.

We report consistent increases in phosphoglycerate dehydrogenase (PHGDH) expression in mouse models of Alzheimers disease (AD) (3xTg-AD) and related tauopathy (PS19), particularly in hippocampal astrocytes. Human single-cell RNA-sequencing data reveal a sequential increase of PHGDH expression in people with no, early, and late AD pathology, which is corroborated by protein mass spectrometry and immunohistochemical analyses of three independent cohorts. A sequential increase of PHGDH expression also correlates with increasing clinical AD symptoms and worsening cognitive decline in patients. The consistent increase of PHGDH expression in six AD cohorts (Mayo, ROSMAP, Mount Sinai, Baltimore, Amsterdam, and UCSD/UCI) corroborates with the recent report of PHGDH extracellular RNA in blood plasma as a candidate biomarker for early diagnosis of AD and offers a caution to the suggested use of L-serine as a potential therapy of AD.

Amyloid plaques are a hallmark neuropathological feature of Alzheimers disease (AD), composed of insoluble amyloid beta (A{beta}) peptide. A{beta} undergoes post-translational modifications that alter their biophysical properties, aggregation kinetics, and neurotoxicity, creating a heterogeneous pool of species that differentially affect AD pathogenesis. Pyroglutamate-modified A{beta} (pEA{beta}) is a particularly aggregation-prone and proteolytically resistant variant that preferentially accumulates within plaque cores, is implicated in early plaque seeding, and is a major target of emerging anti-amyloid immunotherapies. However, the molecular environment surrounding pEA{beta} versus unmodified A{beta} (pan-A{beta}) in the human hippocampus remains incompletely defined. Here, we used Biotinylation by Antibody Recognition (BAR), an in-situ proximity labeling approach, to map and compare the protein-protein interactions (proteomes) of pEA{beta} and pan-A{beta} in formalin-fixed postmortem human hippocampal tissue from pathologically confirmed AD cases and cognitively normal (CN) controls. Differential proteomic analysis identified 48 significantly enriched proteins in AD pEA{beta} captures, 28 in AD pan-A{beta} captures, and 15 in CN pan-A{beta} captures. Whereas no significant enrichment was detected in CN pEA{beta} captures, supporting pEA{beta} as a pathology-associated species. pEA{beta} in AD demonstrated the largest variant-specific signature with 31 unique proteins, pan-A{beta} showed 11 unique proteins in AD, and 14 unique proteins in CN, 16 proteins were shared between AD pEA{beta} and AD pan-A{beta}, with PCSK1N shared across AD pEA{beta}, and AD/CN pan-A{beta}. Pathway enrichment analysis revealed broader biological disruptions linked to pEA{beta}, including synaptogenesis signaling, clathrin-mediated endocytosis, mitochondrial division signaling, and neurotransmitter release. Shared pathways included SNARE signaling, glutamatergic receptor signaling, and netrin signaling. These findings demonstrate that pEA{beta} engages an expanded, variant-specific interactome in human AD hippocampus and designate intracellular trafficking, synaptic signaling, and mitochondrial pathways as network-level vulnerabilities relevant to pEA{beta} pathology in AD. Notably, comparison of CN versus AD pan-A{beta} further distinguished protein networks associated with physiological A{beta} engagement versus pathological pan-A{beta} deposition.

Background and Hypothesis: Alzheimers disease with psychosis (AD+P) is a subgroup of AD patients with more rapid cognitive deterioration. While our previous study showed that AD+P is associated with loss of prefrontal cortex postsynaptic density (PSD) proteins, identifying proteins in the broader cellular environment that influence PSD loss addresses a critical knowledge gap about synaptic dysfunction mechanisms in early disease stages. Study Design: We conducted a proteomic analysis comparing prefrontal grey matter cortex tissue homogenates from elderly normal controls (n=18), individuals with AD+P (n=61), and individuals with AD-P (n=48), all with Braak stages 3-5. Study Results: AD+P showed the most pronounced alterations relative to controls (178 proteins with q<0.05), although alterations in AD-P and AD+P relative to controls were highly similar (R{superscript 2}=0.965, p<0.001). Weighted-gene correlation network analysis (WGCNA) identified four modules significantly associated with disease status comparing AD subjects to controls, but none differed significantly between AD+P and AD-P. We identified 15 proteins significantly correlated with PSD yield across all samples, including ENPP6, linked to AD+P by GWAS. Additionally, PSD yield-associated proteins showed minimal overlap with altered AD proteins (1 of 137). WGCNA revealed one module significantly correlated with PSD yield across all samples, enriched for inflammatory terms. Conclusions: Our findings suggest a model in which AD+P arises from the combination of quantitative alterations within a shared AD proteome profile and a superimposed set of protein alterations correlated with PSD yield that are largely independent of the shared AD proteome, conferring distinct mechanisms of synaptic vulnerability and psychosis risk.

Synaptic dysfunction is an early hallmark of Alzheimers disease, characterized by the disruption of synaptic transmission and plasticity. Central to these processes is endosomal trafficking, mediated by the retromer complex, which orchestrates the movement of vesicle contents for recycling to the plasma membrane, return to the Golgi, or degradation. Variants of VPS35, the cargo recognition component of the retromer complex, have been linked to neurodegenerative diseases, including Parkinsons disease (PARK17, D620N mutation) and Alzheimers disease (L625P mutation). While substantial research has focused on Parkinsons, the role of VPS35 in Alzheimers has been less explored. This study investigates the acute neuroprotective effects of retromer-stabilizing compounds in the 5xFAD mouse model of Alzheimers. Our results reveal that stabilization of the retromer complex not only mitigates pathogenic A{beta} production mechanisms but also compensates for early synaptic dysfunction and microglial activation. Specifically, we observed significant modulation of genes involved in long-term potentiation and a reduction in abnormal retromer-associated cargos. These findings highlight the potential of retromer stabilisation as atherapeutic strategy to address fundamental pathological pathological processes in Alzheimers disease.

According to the prevalent Amyloid Hypothesis, the underlying cause for neurodegeneration in Alzheimer Disease (AD) is attributed to the accumulation of misfolded Amyloid {beta} and tau protein in the form of extracellular sticky plaques and neurofibrillary tangles respectively. These protein accumulations are thought to be caused by impaired waste removal. In an alternative hypothesis, we have proposed the existence of an extensive glial canal system that is likely formed by myelinated aquaporin-4 (AQP4)-expressing tanycytes and removes cellular waste from the hippocampal formation. Here, we demonstrate that tanycyte-derived waste-internalizing receptacles are immunoreactive for A{beta} and emanate from specialized nucleus-like organelles in the following referred to as tanysomes. Utilizing RNA-scope in situ hybridization, we demonstrate that these receptacle-forming tanysomes express RNA for AQP4 and the A{beta}-related genes, amyloid precursor protein, and presenilin 1. These findings suggest that A{beta} is likely synthesized where receptacle formation is observed and that A{beta} may play an important structural role in receptacle formation. In AD-affected hippocampus excessive amounts of A{beta}-immunoreactive waste receptacles emerge from tanysomes and have the appearance of plaques in A{beta}-immunolabeled hippocampus. Moreover, we demonstrate that the same receptacle-forming organelles exhibit strong immunolabeling for hyperphosphorylated tau protein in AD-affected tissue. We postulate that both proteins may play important structural roles in waste uptake and that hypertrophic swelling of impaired tanycytes in AD-affected brain may be due to obstructions of this extensive interconnected glial canal system.

Basal forebrain cholinergic neurons regulate cortical activity and cognition and are vulnerable in Alzheimers disease (AD). However, the upstream circuits controlling projection-defined basal forebrain cholinergic populations remain incompletely understood. Here, we used projection-specific rabies-mediated monosynaptic tracing to map whole-brain inputs to medial prefrontal cortex (mPFC)-projecting cholinergic neurons in the nucleus basalis of Meynert (NBM) and horizontal limb of the diagonal band of Broca (HDB). mPFC-projecting NBM and HDB cholinergic neurons received broad but distinct input patterns. NBM cholinergic neurons received prominent striatal input, including input from D1-expressing medium spiny neurons, whereas HDB cholinergic neurons showed proportionally weaker striatal input and broader non-striatal contributions. Optogenetic electrophysiology confirmed that striatal inputs formed monosynaptic GABAergic inhibitory synapses onto NBM cholinergic neurons. This inhibitory transmission was weakened in 5xFAD mice, indicating impairment of a striatal-NBM inhibitory circuit in an AD mouse model. Together, these findings reveal subregion-specific input organization of mPFC-projecting basal forebrain cholinergic neurons and identify a vulnerable striatal-NBM circuit in AD. HighlightsO_LIWhole-brain rabies tracing reveals input organization of mPFC-projecting BF cholinergic neurons. C_LIO_LINBM and HDB cholinergic neurons projecting to mPFC show distinct monosynaptic input profiles. C_LIO_LIStriatal D1-MSNs are a major input source to mPFC-projecting NBM cholinergic neurons. C_LIO_LIStriatal-NBM inhibitory transmission is functionally impaired in 5xFAD mice. C_LI

Millions of people suffer worldwide from neurodegenerative diseases ranging from rapidly progressing and fatal motor neuron diseases like Amyotrophic Lateral Sclerosis (ALS) to more chronic illnesses such as frontotemporal dementia (FTD) and Alzheimers disease (AD). A growing number of studies have implicated neuroinflammation as a key and causative phenomenon and an important target for novel therapeutics for these diseases. Neuroinflammation is characterized by reactive glial cells that produce pro-inflammatory neurotoxic cytokines. Our previous studies have shown a brain-penetrant Nanoligomer cocktail (NI112) inhibiting the neuroinflammation mediators nuclear factor kappa-light-chain-enhancer of activated B cells (NF-{kappa}B) and NOD-like receptor family, pyrin domain containing 3 (NLRP3) is a safe, targeted, and effective neurotherapeutic drug. Here, we show that a four-week NI112 treatment is therapeutic using: 1) an ALS-FTD 3D human motor neuron organoid model of tar DNA binding protein 43 (TDP-43, a key contributor to ALS pathology) overexpression (knock-in); 2) an AD model of APOE4/APOE4 (AD risk allele) double mutation in human neurons comprising a 3D human prefrontal cortex (PFC) organoid; and 3) multiple in vivo (mouse models) of the same/related conditions. In 3D organoids made from healthy motor neurons (HMN negative control) and TDP-43 overexpressing (or ALS organoids), we monitored the mean firing rate using calcium signaling as a functional output, while measuring TDP-43 and other key neurodegeneration biomarkers. After 4 weeks, we observed a massive improvement in the mean firing rate of NI112-treated ALS organoids compared to untreated ALS organoids, which was more comparable to healthy HMN organoids. Similarly, we found a significant decrease in neurodegeneration markers like amyloid beta 42 (A{beta}42) in NI112-treated AD organoids compared to untreated AD organoids (A{beta}42 comparable to healthy PFC organoids). In the mouse ALS (SOD1-G93A) model, we observed behavioral improvements and restoration of motor function (e.g., grip strength) in NI112-treated mice, and in mouse AD model mice (radiation-induced accelerated neuropathology in APP/PS1, and rTg4510 phospho-tau), we observed improved cognition. In both models, we also found an accompanying reduction in neuroinflammation and reduced neuropathology. These results show the promise for further testing and development of neuroinflammation-targeting Nanoligomers to benefit patients suffering from debilitating neurodegenerative diseases like ALS, FTD, and AD.

BackgroundMitochondrial dysfunction is an early and central feature of Alzheimers disease (AD). In particular, intercellular mitochondrial transfer has emerged as a mechanism of neuronal support in brain injury and neurodegeneration. However, pathways governing astrocyte-to-neuron transfer and its role in AD pathogenesis remain unknown. MethodsUsing the AppNL-G-F knock-in AD model, we combined high-resolution 4D live-cell imaging with quantitative fluorescence-based reporters to assess synaptic function and mitochondrial network dynamics in neurons and astrocytes. Direct and extracellular vesicle (EV)-restricted neuron-astrocyte co-culture systems were used to investigate bidirectional mitochondrial transfer. We performed the first in-depth structural, proteomic, and functional characterization of astrocyte-derived mitochondrial extracellular vesicles (mitoEVs) using cryo-electron microscopy, quantitative mass spectrometry, and bioenergetic analyses to define their cargo composition and metabolic effects. ResultsWe identified cell-type-specific mitochondrial remodeling in early AD, with compartmentalized synaptic energy deficits in neurons and hyperdynamic, less interconnected, yet metabolically preserved networks in astrocytes, preceding global bioenergetic decline. Bidirectional mitochondrial transfer between astrocytes and neurons, also at axonal terminals, was mediated by specialized mitoEVs but significantly reduced in the AppNL-G-Fmodel. Comprehensive proteomic and functional profiling revealed that WT astrocyte-derived mitoEVs are enriched in inner membrane and matrix proteins, supporting oxidative phosphorylation, lipid and amino acid metabolism, and redox homeostasis. In contrast, AppNL-G-F mitoEVs are selectively depleted of respiratory and fatty acid oxidation components and exhibit impaired respiration with reduced Complex IV activity. Functionally, WT mitoEVs promote mobilization of abnormal accumulation of lipid droplets in AppNL-G-Fneurons, restore fatty acid oxidation, and increase neuronal bioenergetics, including at the synapses. In contrast, disease-derived mitoEVs fail to engage these pathways. ConclusionsTogether, these findings identify mitoEV-mediated mitochondrial transfer as a glia-to-neuron metabolic pathway compromised in early AD and reveal a coordinated role for oxidative phosphorylation and fatty acid oxidation in supporting synaptic energy homeostasis.

-Synuclein nitration is a prominent post-translational modification in Parkinsons disease, but whether nitrated -synuclein merely reflects oxidative stress or actively contributes to pathology remains unclear. Here, we generated and characterized 6G6, an antibody selective for Tyr39-nitrated -synuclein, and tested whether targeting this modified -synuclein species affected pathology in different mouse models of -synuclein aggregation and spread. In two models of -synuclein overexpression targeting medullary vagal neurons, oxidative stress was induced by either exposure to the herbicide paraquat or transgenic heterozygous expression of the Gba1-L444P mutation. Both conditions were characterized by robust -synuclein spreading that was markedly counteracted by 6G6 administration. A third model consisted of an injection of -synuclein fibrils into the striatum of -synuclein-overexpressing mice. In this model, treatment with 6G6 protected against fibril-induced aggregate pathology and ensuing degeneration of nigral dopaminergic neurons. In a pilot human study, CSF levels of Tyr39-nitrated -synuclein were measured and found increased in Parkinson patients as compared to controls. These findings identify Tyr39-nitrated -synuclein as a pathogenic, therapeutically targetable -synuclein species linking oxidative/nitrative stress to PD pathological processes.

Analyzing Alzheimers disease (AD) pathology within anatomical subregions is a significant challenge, often carried out by pathologists using a standardized, semi-quantitative approach. To augment traditional methods, a high-throughput, high-resolution pipeline was created to classify the distribution of AD pathology within hippocampal subregions. USC ADRC post-mortem tissue sections from 51 patients were stained with 4G8 for amyloid, Gallyas for neurofibrillary tangles (NFTs) and Iba1 for microglia. Machine learning (ML) techniques were utilized to identify and classify amyloid pathology (dense, diffuse and APP (amyloid precursor protein)), NFTs, neuritic plaques and microglia. These classifications were overlaid within manually segmented regions (aligned with the Allen Human Brain Atlas) to create detailed pathology maps. Cases were separated into low, intermediate, or high AD stages. Further data extraction enabled quantification of plaque size and pathology density alongside ApoE genotype, sex, and cognitive status. Our findings revealed that the increase in pathology burden across AD stages was driven mainly by diffuse amyloid. The pre and para-subiculum had the highest levels of diffuse amyloid while NFTs were highest in the A36 region in high AD cases. Moreover, different pathology types had distinct trajectories across disease stages. In a subset of AD cases, microglia were elevated in intermediate and high compared to low AD. Microglia also correlated with amyloid pathology in the Dentate Gyrus. The size of dense plaques, which may represent microglial function, was lower in ApoE4 carriers. In addition, individuals with memory impairment had higher levels of both dense and diffuse amyloid. Taken together, our findings integrating ML classification approaches with anatomical segmentation maps provide new insights on the complexity of disease pathology in AD progression. Specifically, we identified diffuse amyloid pathology as being a major driver of AD in our cohort, regions of interest and microglial responses that might advance AD diagnosis and treatment.

Alzheimers disease (AD) is characterized by early synaptic dysfunction followed by progressive amyloid-{beta} (A{beta}) accumulation, neuroinflammation, and cognitive decline. We previously identified Secreted Frizzled-Related Protein 1 (SFRP1) as a multifactorial contributor to AD pathogenesis and provided initial evidence that its neutralization ameliorates pathological AD-like traits in mice. Here, we evaluate the pharmacodynamics, biodistribution, and therapeutic window of an -SFRP1 monoclonal antibody (-SFRP1) in APP/PS1 mice. Pharmacokinetics and target engagement of -SFRP1 were assessed in different groups of APP/PS1 mice using biotinylated or 89Zr-labelled antibodies, with tissue distribution and -SFRP1 levels quantified by in-house ELISA or PET/CT. Therapeutic efficacy was evaluated by administering -SFRP1 or the SFRP1 inhibitor WAY-316606 at different stages of disease progression via retro-orbital injection, followed by analysis of AD-like pathology using ELISA and immunofluorescence assays followed by quantifications and statistical analysis. Using 89Zr-labelled antibodies, we show that intravenously administered -SFRP1 engages its target systemically and reaches the brain, although at substantially lower levels and with a rapid 24-hour clearance. Treatment with -SFRP1 had no apparent systemic side effects, but its therapeutic efficacy against AD-like brain pathology was strongly dependent on disease stage. While early administration reduced amyloid pathology in previous studies, treatment initiated at intermediate or advanced stages showed minimal benefit at standard doses. Higher antibody doses reduced amyloid burden and dystrophic neurites but were associated with increased mortality with time. Pharmacological inhibition of SFRP1 using a small-molecule inhibitor similarly failed to ameliorate pathology at intermediate stages. Together, these findings demonstrate that SFRP1 remains a relevant therapeutic target in AD, but its effective modulation is constrained by limited brain exposure and a narrow therapeutic window, underscoring the importance of early intervention and prompting the search for improved brain-targeted delivery strategies.

Alzheimers disease (AD) is defined by the accumulation of neurofibrillary tangles containing hyperphosphorylated Tau and plaques containing Amyloid-{beta} (A{beta}). The aggregation of these two proteins is considered central to the disease. The lack of animal models that can recapitulate A{beta} and tau pathologies without overexpressing these proteins has hindered AD research. Accelerating pathology by inoculating A{beta} and tau seeds has helped to understand their prion-like propagation in the brain. Previous studies failed to characterise both A{beta} and tau pathologies in vivo upon inoculating AD brain homogenates. Here we present a longitudinal and systematic study; we inoculated the AppNL-F/NL-F knockin mice, which express humanised A{beta} and murine wild-type tau, with extracts from diseased human brains to analyse the contribution of A{beta} and tau assemblies to AD pathogenesis. We found that mice inoculated with AD brain extracts evinced early and prominent amyloid deposition, while those injected with control brain extracts or vehicle did not. Parenchymal and vascular amyloid accumulated in the same brain regions affected in control-inoculated AppNL-F/NL-F mice. However, the extent of vascular amyloid far exceeded that seen in AppNL-F/NL-Fmice injected with control brain extracts, and parenchymal deposits extended to a previously untargeted brain region - the cerebellum. An end-point titration of an AD brain homogenate in AppNL-F/NL-F mice demonstrated that human A{beta} seeds can be titrated in a prion-like fashion, which is useful for sample comparison, diagnostic and risk studies. Notably, the inoculation of AppNL-F/NL-F mice with AD brain homogenate induced intense tau phosphorylation, and provides more detailed context for the inoculation of AppNL-F/NL-F mice with human samples to study temporal and mechanistic relationships between A{beta} and tau pathology, vascular amyloid deposition and bioactivity of A{beta} seeds.